Editing NGS Library Preparation (section)

==== Illumina RNA-Seq Library Preparation. ====

Total RNA quantity and integrity were assessed using Quant-IT RiboGreen RNA Assay Kit (Invitrogen, Carlsbad, CA, USA) and Agilent Tapestation 2200 R6K. Libraries for Illumina sequencing were constructed from 100 ng of total RNA using the NEBNext mRNA Sample Prep Kit 1 (New England Biolabs, Ipswich, MA, USA), following the manufacturers' guidelines with minor modifications: end repair in 50 µl reaction volume (40 µl DNA, 5 µl buffer and 5 µl enzyme), post fragmentation clean-up with 1:2.8× volume Agencourt Ampure RNAClean XP (Beckman Coulter, Pasadena, CA, USA); post cDNA synthesis clean-up with 1:1.25× volume Ampure XP Beads; post end repair, A-Tailing and adapter ligation clean-ups with 1:1.8× volume Ampure XP Beads and post-PCR library clean-ups with 1:1× volume Ampure XP Beads. Additionally, upon ligation of Illumina Adapters (Multiplexing Sample Preparation Oligonucleotide Kit) each library was size selected with two Ampure Bead steps (firstly, 1:0.7× volume and secondly, the supernatant from the first bind was taken for a 1:1.7× volume clean-up), selecting 200–600 bp fragments in 30 µl 10 mM Tris-Cl, pH 8.5. Pre-PCR workflow was partially performed using a Beckman Biomek FX and post-PCR steps were performed using a Beckman Biomek NX<sup>P</sup> and Biomek 3000.



The following custom primers (25 µM each) were used for the PCR enrichment step:



Multiplex PCR primer 1.0



<span class="gene">5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′</span>



Index primer



<span class="gene">5′-CAAGCAGAAGACGGCATACGAGAT</span>[INDEX]<span class="gene">CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′</span>



Amplified libraries were analysed for size distribution using the Agilent Tapestation 2200 D1K. Libraries were quantified by quantitative RT-PCR using Agilent qPCR Library Quantification Kit and a Mx3005P instrument (Agilent) and relative volumes were pooled accordingly. Finally, a second quantitative RT-PCR was performed to measure the relative concentration of the pool compared to a previously sequenced mRNA library in order to determine the volume to use for sequencing.